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An electrophoretic and immunologic study of Murex trunculus haemocyanin
Comp. Biochem. PhydoL, 1968, Vo/. 26, pp. 345 to 351. Pergamon Press. Printed in Great Britain
AN ELECTROPHORETIC AND IMMUNOLOGIC STUDY OF M U R E X TRUNCULUS HAEMOCYANIN E. J. W O O D , C A T H E R I N E M. SALISBURY, N. F O R M O S A and W. H. B A N N I S T E R Department of Physiology and Biochemistry, Royal University of Malta, Valletta, Malta (Received 18 jYanuary 1968) A b s t r a c t - - 1 . Rabbit antiserum to Murex trunculus haemocyanin gave a single precipitation line in agar diffusion against M . trunculus haemocyanin and apohaemocyanin, and gave a reaction of partial identity with M . brandaris
haemocyanin. 2. A single precipitation arc appeared in microimmunoelectrophoresis at pH 6.0-9.2. If EDTA was present, one arc was produced at pH 6"4, but two arcs appeared at pH 8"15 indicating dissociation into subunits. 3. In cellulose acetate membrane electrophoresis one band appeared at pH 5.4-7.3, and four to six bands were found at pH 8.15-10.8. Calcium or magnesium ions prevented the appearance of multiple bands at pH 8"15 and 9.2. INTRODUCTION MUCH work on the haemocyanins (see Ghiretti, 1962, 1956) has revealed that they are largely similar in many properties, although haemocyanins from different species are in detail quite distinct. A broad division is recognizable between the low molecular weight arthropod haemocyanins and the high molecular weight molluscan haemocyanins, but even among quite closely related members of either group, there are minor differences in amino acid composition and copper content, and pronounced differences in the number of free sulphydryl groups (GhirettiMagaldi & Nuzzolo, 1965, Ghiretti-Magaldi et al., 1955). Nevertheless, the functional properties of all haemocyanins, that is, their ability to transport oxygen, are very similar. This presumably means that the combining sites are all very similar, whereas the rest of the protein molecule shows the normal sort of differences that go to make up species specificity. It is known from ultracentrifuge (van Holde & Cohen, 1964), light-scattering (Elliott & van Baelen, 1965), and electron microscope studies (Fern~ndez-Mor~n et al., 1966) that haemocyanins are capable of splitting into subunits, usually halves, and then eighths, tenths or twelfths, depending on the pH and on the presence or absence of certain ions, notably Ca 2+ and Mg a+. It is also possible to demonstrate this dissociation by means of electrophoresis (Elliott & van Baden, 1965). In the present work some of the immunological and electrophoretic properties of the haemocyanin from the whelk, M u r e x trunculus (L.), have been examined. 345
346
E.J. WOOD,CATHERINEM. SALISBURY,N. FORMOSAAND W. H. BANN,STER
MATERIALS AND M E T H O D S Haemocyanins M. trunculus haemoeyanin was prepared from haemolymph by the method of Bannister et al. (1966). The copper content of the freshly purified material was determined by the method of Peterson & Bollier (1955), and was found to be within the range 0.24--0.26%. M. trunculus apohaemocyanin was prepared by removing the copper with cyanide according to the method given by Cohen & van Holde (1964). Photooxidized haemocyanin was prepared as described previously (Wood & Bannister, 1968) except that the temperature during the reaction was 24°C. After 85 rain irradiation, 7.2/~mole oxygen had been consumed and about 50 per cent of the copper had been lost. Haemolymph was obtained from Murex brandaris (L.), Cerithium vulgatum (Brug.) and Euthria cornea (L.), by removing the tip of the shell with bone forceps and inverting the shell over a small funnel whereupon haemolymph drained from the wound. The haemolymph was centrifuged to remove shell and tissue debris and then stored under toluene.
Preparation of antiserum to M. trunculus haemocyanin Purified M. trunculus haemocyanin was dialysed against several changes of 0"85% (w/v) NaC1, and then diluted with the same saline to a final concentration of 20 mg/ml. The solution was sterilized by passage through an HP]EKS Seitz pad (Carlson-Ford Ltd., Ashton-under-Lyne, Lancashire). Five rabbits weighing about 2 kg received six intradermal injections of 0.5 ml (10 mg) haemocyanin over 2 weeks. Test bleedings taken from the marginal ear vein 1 week after the last injection gave a strong reaction against M. trunculus haemocyanin. The rabbits were therefore anaesthetized with Nembutal (Abbott Laboratories Ltd., London) and exsanguinated. The serum was separated from cells and clot, and the pooled sera were preserved by means of thiomersalate (1 : 10,000). Electrophoresls, immunodiffusion, and microimmunoelectrophoresis Electrophoresis on cellulose acetate membranes (Oxoid Ltd., London) was performed in a Shandon-Kohn apparatus. The strips were stained in 0"2% (w/v) Ponceau S containing 3 % (w/v) trichloroaeetic acid. Microimmunoelectrophoresis was performed on microscope slides in 1% Ionagar (Oxoid) made up in the appropriate buffer, in the same electrophoresis tank. Agar-gel immunodiffusion was performed in the same agar on 2 x 3-in. microscope slides. These gels, and the microimmunoelectrophoresis gels were stored in a moist atmosphere at room temperature and inspected at intervals over 1--4 days. RESULTS
Immunological reactions of haemocyanins As was to be expected f r o m previous reports (Garvey et al., 1967) M . trunculus haemoeyanin, like other haemocyanins, was a good antigen in rabbits. All five rabbits produced a strong antibody to haemocyanin after the immunization schedule described above. I n agar-gel diffusion a single precipitation line was produced with M . trunculus haemocyanin, and the corresponding apohaemoeyanin reacted similarly. O f the other haemoeyanins tested, that of M . brandaris gave a reaction of partial identity (Fig. 1), while those of C. vulgatum and E. cornea failed to react. I n microimmunoelectrophoresis M . truneulus haemocyanin, as well as the corresponding apohaemocyanin, gave a single precipitation are at all p H values tested between 6-0 and 9-6. T h e mobilities of haemoeyanin and apohaemocyanin appeared to be identical. Some small variations in the pattern obtained were observed depending on the age of the haemoeyanin preparation and on its previous
MUREX TRUNCULUS HAEMOCYANIN
347
treatment. Thus, fresh M . trunculus haemolymph or freshly purified haemocyanin gave a single sharp arc, whereas haemocyanin that had been lyophilized in the presence of sucrose (Heirwegh et al., 1959) and then stored for several months, showed small spurs at either end of the precipitation arc. In all cases however this effect was very small. Photooxidized haemocyanin reacted with the antiserum (Fig. 2D, E) but had a lower mobility than native haemocyanin. Cellulose acetate membrane electrophoresis
Haemocyanin was dialysed against buffers of pH values from 5.4 to 10.8, and then electrophoresis was performed in the same buffer. At pH values below about
All
0
•
!
Fio. 3. Cellulose acetate membrane electrophoresis of M. trunculus haemocyanin at different pH values (tracing). A, 0"1 M acetate, pH 5"5, 100 rain; B, 0.04 M veronal, pH 7"3, 80 rain; C, 0"04 M veronal, pH 8.3, 90 min; D, 0.04 M veronal, pH 9.1, 90 rain; E, 0"2 M glycine, pH 10"4, 90 min. 7.3 a single band was observed, whereas at pH values of 8-3 and above, a number of bands, usually four to six, was always obtained. As Fig. 3 shows, the most common pattern was two strong bands, preceded and followed by weaker bands. In all cases movement was towards the anode. Of the other haemocyanins tested, M . brandaris haemocyanin gave a single band at pH 7.0 which moved slightly more slowly than that of M . trunculus (ratio of mobilities, 1 : 0.93), and three to four bands at pH 9.2. In the latter case a very similar pattern to that produced by M . trunculus haemocyanin was observed. E. cornea haemocyanin similarly produced one band at pH 7.0 and three bands at pH 9.2. At pH 7.0 apohaemocyanin from M . trunculus produced a single band of about the same mobility as native haemocyanin, but there was a faint smear extending back to the origin. At pH 8.6 apohaemocyanin produced two to three diffuse bands, in about the same positions as the bands produced by native haemocyanin, but
348
E.J.
WOOD,
CATHERINEM.
SALISBURY,
N. FORMOSAANDW. H. BANNISTER
again there was streaking back to the origin. Photooxidized haemocyanin produced a smear extending from the origin at all pH values, and no bands could be identified.
Effect of Ca 2+ and Mg 2+ on the stability of haemocyanin In the experiments described above, the observed behaviour is presumably an expression of the tendency of haemocyanins to dissociate into subunits depending on the pH (see Discussion). In these experiments the haemocyanin or haemolymph had been dialysed against 0.1 M phosphate buffer, and was then dialysed against the buffer to be used during electrophoresis. The results therefore show the electrophoretic behaviour of haemocyanin in the calcium- and magnesium-free state. It is known that calcium and magnesium ions are capable of stabilizing haemocyanins from other species against this process of dissociation (Elliott & van Baelen, 1965 ; van Holde & Cohen, 1964). For example, whereas in the absence of Ca 2+ or Mg z+, Helix pomatia haemocyanin starts to dissociate into subunits at pH 7-4-8.1, in the presence of either of these ions, dissociation does not occur until a pH above 9 is reached (Lontie & Witters, 1966; Lontie et al., 1962). Experiments were therefore performed in which haemocyanin was dialysed overnight at 4°C against buffers at various pH values which contained calcium or magnesium salts, and then subjected to electrophoresis in the same buffer. The results are set out in Table 1. Both Ca 2+ and Nlg 2+ at 10 mM concentration extended the stability TABLE 1--EFFECT OF Ca AND Mg
IONS ON M . Trunculus HAEMOCYANIN IN CELLULOSE ACETATE MEMBRANE ELECTROPHORESIS
Dialysis and electrophoresis
Result
pH 7-0 phosphate pH 7"0 phosphate + 10 mM Ca2+ pH 8"15 veronal pH 8"15 veronal + 10 mM Ca2+ pH 8"15 veronal + 10 mM Mg~+ pH 9"2 veronal pH 9"2 veronal+ 10 mM Ca2+ pH 9.2 veronal + 10 rnM Mg2+ pH 9-2 veronal + 100 mM Mg2+
1 band 1 band 4-5 bands 1 band 1 band 4-5 bands lband 3 bands 1 band
region of M. trunculus haemocyanin to pH 8.15 so that a single band was produced. At pH 9.2, Mg ~+ appeared to be less effective in stabilizing the macromolecular structure than Ca ~+, since 10 mM Ca z+, but 100 mM Mg ~+ were required to give a single band at this pH. So far we have been unable to reverse the process of dissociation into subunits in the absence of Mg 2+ or Ca 2+. If haemocyanin was brought to pH 9.2 in the absence of these ions, when it produced four to five bands in electrophoresis, and then dialysed at pH 7.0 either in the presence or absence of calcium or magnesium salts, four to five bands were still produced in electrophoresis at pH 7"0.
M U R E X TRUNCULUS HA.EM0CYANIN
349
No evidence of any heterogeneity was found in microimmunoelectrophoresis under normal conditions over the pH range 6.0-9.6. However, since the agar used contained 0.28% Ca and 0.32% Mg (The Oxoid Manual), it seemed likely that there was enough present of either of these ions to prevent dissociation of haemocyanin in the alkaline pH range. To obviate any effect of these ions, experiments were performed with buffers containing 50 mM EDTA. At pH 6"4 (Fig. 2G) a single precipitation arc formed, but at pH 8.15 (Fig. 2F) two arcs were produced indicating the existence of two species of different mobility and antigenic composition under these conditions. DISCUSSION The present work demonstrates that M. truneulus haemocyanin, like other haemocyanins, is a good antigen, that it reacts in immunodiffusion with a specific antiserum to give a single precipitin line and that in immunoelectrophoresis at pH values below 7"4 in the presence of Ca ~+ or Mg 2+, it gives a single precipitation arc. Furthermore, lyophilization in the presence of sucrose resulted in very small changes as judged by these immunological criteria. The rabbit antiserum to M. trunculus haemocyanin gave a reaction of partial identity with the haemocyanin of the related species M. brandaris, but did not react with the haemocyanins from two other gastropods, E. cornea and C. vulgatum. Malley et al. (1965) demonstrated that the haemocyanin of the giant keyhole limpet (Megathura crenulata) and that of the horseshoe crab (Limulus polyhemus) had no common antigenic determinants. Similarly, Parisi et al. (1962) found that Homarus, Eriphia and Xiphosura haemocyanins had no common determinants. The apohaemocyanin prepared from M. trunculus haemocyanin by treatment with cyanide reacted identically to native haemocyanin. In a study of some of the immunological properties of haemocyanins, Ghiretti's group (Parisi et al., 1962) found that fresh Octopus haemocyanin gave a single sharp precipitation line with its antiserum in immunoelectrophoresis, but that the corresponding apohaemocyanin produced a diffuse arc. They also showed that the precipitation arc produced by apohaemocyanin did not contain copper, and thus did not represent a small amount of contaminating native haemocyanin. It seems therefore that removal of copper by means of cyanide, particularly in the case of M. trunculus haemocyanin, causes minimal changes in the conformation of the protein, which are insufficient to affect the antigenic sites. It is notable that photooxidation of the histidine (and probably other) residues of haemocyanin does not destroy the antigenic reaction. This is similar to the finding of Finger & Schaeg (1967), that complete destruction of all the histidine of insulin left a considerable amount of antigenicity. That the photooxidized insulin and haemocyanin had altered electrophoretic mobilities was to be expected from the destruction or modification of polar residues. Here again one may conclude that destruction of histidine residues in these proteins by means of photooxidation in the presence of methylene blue, does not lead to gross changes in conformation. This is important in the case of haemocyanin, since photooxidation causes loss of
350
E.J. WOOD, CATHERINEM. SALISBURY,N. FORMOSAAND W. H. BANNISTER
copper (Wood & Bannister, 1968), while it is known at the same time that denaturing agents (acids, alcohol, urea) can also cause loss of copper, simply by disrupting the conformation of the protein (see Ghiretti, 1962; Griffd & Lontie, 1961). Previous studies of the reaction of haemocyanin and antiserum in immunoelectrophoresis and in immunodiffusion appear to have taken no account of the calcium and magnesium content of the agar. Parisi et al. (1962) used 1% agar of unspecified origin in a pH 8.4 veronal buffer, and Malley et al. (1965) used 0.85% Ionagar No. 2 (Consolidated Laboratories, Inc.) in pH 8"6 borate buffer. In view of the present findings in experiments where E D T A was included in the buffer, it is perhaps fortuitous that single precipitation arcs were obtained in this previous work, especially as the pH values were beyond the stability zones of some of the haemocyanins. The alternative possibility is that the haemocyanins were completely dissociated into the smallest subunits under these conditions. This is probably less likely, although it does depend on the stability region of the haemocyanin in question. The present finding with cellulose acetate membrane electrophoresis has demonstrated that M. trunculus haemocyanin is heterogeneous in electrophoresis at certain pH values depending upon the presence or absence of calcium or magnesium (Table 1). It seems probable that this may be attributed to subunit formation and one may conclude that the subunits formed are not identical. It is known from ultracentrifuge studies (R. Lontie, personal communication) that M. trunculus haemocyanin splits into half, and then eighth-to-twelfth molecules with increasing pH. However, since between four and six bands were obtained in electrophoresis at pH values above 7.3, it appears likely that either two dissimilar half molecules are produced, or that the smaller subunits are dissimilar, or both. Since a single component appears in the ultracentrifuge at pH values greater than about 8.4 (R. Lontie, personal communication), it seems that small subunits of equal size but of different charge are produced. In cellulose acetate membrane electrophoresis Elliott & van Baelen (1965) found that the haemocyanin from the Congo river snail (Pila le@oldvillensis) gave a single band at pH 5.47, two bands at pH 7.25, three to four bands at pH 8.35, and a single band at pH 9.05. They concluded that the bands obtained represented subunits, and they confirmed this by light-scattering experiments in which the molecular weight was determined as a function of pH. In the buffer of the highest pH (9.05), therefore, a single small subunit exists in Pila haemocyanin. We have been unable to obtain a single band at any pH above 7.3 with M. trunculus haemocyanin. Even in experiments at pH 10.8 several bands continued to appear. REFERENCES
BANNISTERW. H., BANNISTERJ. V. & MICALLEFH. (1966) Purification of hemocyanin from hemolymph by adsorption to calcium phosphate. Experientia 22, 626-627. COHENL. B. & VANHOLDEK. E. (1964) Physical studies of hemocyanins--II. A comparison of the hemocyanin and apohemocyanin of Loligo pealei. Biochemistry 3, 1809-1813. ELmOTT F. G. & VANBAELENH. (1965) Poids moldculaire et zone de stabilitd de l'hdmocyanine de Pila leopoldvillensis. Bull. Soc. Chin. biol. 47, 1979-1986.
MU.R.EX T R U N C U L U S HAEMOCYANIN
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F ~ L , CDEz-MoR~ H., VAN BRUGGEN E. F. G. & OHTSUKI M. (1966) Macromolecular organization of hemocyanins and apohemocyanins as revealed by electron microscopy. jT. Molec. Biol. 16, 191-207. FINGER H. & SCHA~GW. (1967) Die Antigeni~t des photooxidierten Insulins. Biochim. biophys. Acta 140, 532-534. GARWYJ. S., CAMPBELLD. H. & DAS M. L. (1967) Urinary excretion of foreign antigens and RNA following primary and secondary injections of antigens, ft. exp. Med. 125, 111-126. GHIR~tTI F. (1962) Hemerythrin and hemocyanin. In Oxygenases (Edited by HAYAISHIO.), pp. 517--553. Academic Press, New York. GHIlmTTI F. (1966) Molluscan hemocyanins. In Physiology of Mollusca (Edited by WILBUR K. M. & YONGEC. M.), Vol. 2, pp. 233-248. Academic Press, New York. GHImn~rI-MAGALDXA. & NuZZOLOC. (1965) Thiol groups in hemocyanins. Comp. Biochem. Physiol. 16, 249-252. GHIRETTI-MAOALDIA., NUZZOLOC. & GHII~TTI F. (1966) Chemical studies on hemocyanins - - I . Amino acid composition. Biochemistry 5, 1943-1951. G~IFF[ M. & LONTm R. (1961) L'action de l'ur~e sur la liason du cuivre dans l'h6mocyanine d'Helix pomatia. Archs int. Physiol. Biochim. 69, 594--595. HEXRWEGHK., BORGINONH. & LONTm R. (1959) Sur la possibilit~ de lyophiliser les h~mocyanines d'Helix pomatia en pr6sence de saccharose. Archs int. Physiol. Biochim. 67, 514--516. LONTm R., BRAUNSG., COOReMANH. & VANCLEFA. (1962) Distribution of c~- and flhemocyanins in the blood ofHelixpomatia..drchs Biochem. Biophys. Suppl. 1,295-300. LONTIER. & WITTERSR. (1966) Helixpomatia hemocyanins. In The Biochemistry of Copper (Edited by I~mACHJ., AISF~ P. & BLUMBERGW. E.), pp. 455--463. Academic Press, New York. MALLEYA., SAHAA. & HALLIDAYW. J. (1965) Immunochemical studies of haemocyanins from the giant keyhole limpet (Megathura crenulata) and the horseshoe crab (Limulus polyhemus). .7. Immunol. 95, 141-147. PARXSIV., NARDXG., GHIRETTIF. & GHIRET'rx-MACALDXA. (1962) Ricerche sulle emocianine - - I I I . Proprieta' immunologiche. Boll. Soc. ital. Biol. sper. 38, 1848-1851. PETERSONR. E. & BOLLIERM. E. (1955) Spectrophotometrlc determination of serum copper with biscyclohexanoneoxalyldihydrazone. Analyt.Chem. 27, 1195-1197. VAN HOLDEK. E. & COHENL. B. (1964-) Physical studies of hemocyanins--I. Characterization and subunit structure of Loligo pealei hemocyanin. Biochemistry 3, 1803-1808. WOOD E. J. & BANNISTERW. H. (1968) The effect of photooxidation and histidine reagents on Murex trunculus haemocyanin. Biochim. biophys. Acta 154, 10-16.
AN ELECTROPHORETIC AND IMMUNOLOGIC STUDY OF M U R E X TRUNCULUS HAEMOCYANIN E. J. W O O D , C A T H E R I N E M. SALISBURY, N. F O R M O S A and W. H. B A N N I S T E R Department of Physiology and Biochemistry, Royal University of Malta, Valletta, Malta (Received 18 jYanuary 1968) A b s t r a c t - - 1 . Rabbit antiserum to Murex trunculus haemocyanin gave a single precipitation line in agar diffusion against M . trunculus haemocyanin and apohaemocyanin, and gave a reaction of partial identity with M . brandaris
haemocyanin. 2. A single precipitation arc appeared in microimmunoelectrophoresis at pH 6.0-9.2. If EDTA was present, one arc was produced at pH 6"4, but two arcs appeared at pH 8"15 indicating dissociation into subunits. 3. In cellulose acetate membrane electrophoresis one band appeared at pH 5.4-7.3, and four to six bands were found at pH 8.15-10.8. Calcium or magnesium ions prevented the appearance of multiple bands at pH 8"15 and 9.2. INTRODUCTION MUCH work on the haemocyanins (see Ghiretti, 1962, 1956) has revealed that they are largely similar in many properties, although haemocyanins from different species are in detail quite distinct. A broad division is recognizable between the low molecular weight arthropod haemocyanins and the high molecular weight molluscan haemocyanins, but even among quite closely related members of either group, there are minor differences in amino acid composition and copper content, and pronounced differences in the number of free sulphydryl groups (GhirettiMagaldi & Nuzzolo, 1965, Ghiretti-Magaldi et al., 1955). Nevertheless, the functional properties of all haemocyanins, that is, their ability to transport oxygen, are very similar. This presumably means that the combining sites are all very similar, whereas the rest of the protein molecule shows the normal sort of differences that go to make up species specificity. It is known from ultracentrifuge (van Holde & Cohen, 1964), light-scattering (Elliott & van Baelen, 1965), and electron microscope studies (Fern~ndez-Mor~n et al., 1966) that haemocyanins are capable of splitting into subunits, usually halves, and then eighths, tenths or twelfths, depending on the pH and on the presence or absence of certain ions, notably Ca 2+ and Mg a+. It is also possible to demonstrate this dissociation by means of electrophoresis (Elliott & van Baden, 1965). In the present work some of the immunological and electrophoretic properties of the haemocyanin from the whelk, M u r e x trunculus (L.), have been examined. 345
346
E.J. WOOD,CATHERINEM. SALISBURY,N. FORMOSAAND W. H. BANN,STER
MATERIALS AND M E T H O D S Haemocyanins M. trunculus haemoeyanin was prepared from haemolymph by the method of Bannister et al. (1966). The copper content of the freshly purified material was determined by the method of Peterson & Bollier (1955), and was found to be within the range 0.24--0.26%. M. trunculus apohaemocyanin was prepared by removing the copper with cyanide according to the method given by Cohen & van Holde (1964). Photooxidized haemocyanin was prepared as described previously (Wood & Bannister, 1968) except that the temperature during the reaction was 24°C. After 85 rain irradiation, 7.2/~mole oxygen had been consumed and about 50 per cent of the copper had been lost. Haemolymph was obtained from Murex brandaris (L.), Cerithium vulgatum (Brug.) and Euthria cornea (L.), by removing the tip of the shell with bone forceps and inverting the shell over a small funnel whereupon haemolymph drained from the wound. The haemolymph was centrifuged to remove shell and tissue debris and then stored under toluene.
Preparation of antiserum to M. trunculus haemocyanin Purified M. trunculus haemocyanin was dialysed against several changes of 0"85% (w/v) NaC1, and then diluted with the same saline to a final concentration of 20 mg/ml. The solution was sterilized by passage through an HP]EKS Seitz pad (Carlson-Ford Ltd., Ashton-under-Lyne, Lancashire). Five rabbits weighing about 2 kg received six intradermal injections of 0.5 ml (10 mg) haemocyanin over 2 weeks. Test bleedings taken from the marginal ear vein 1 week after the last injection gave a strong reaction against M. trunculus haemocyanin. The rabbits were therefore anaesthetized with Nembutal (Abbott Laboratories Ltd., London) and exsanguinated. The serum was separated from cells and clot, and the pooled sera were preserved by means of thiomersalate (1 : 10,000). Electrophoresls, immunodiffusion, and microimmunoelectrophoresis Electrophoresis on cellulose acetate membranes (Oxoid Ltd., London) was performed in a Shandon-Kohn apparatus. The strips were stained in 0"2% (w/v) Ponceau S containing 3 % (w/v) trichloroaeetic acid. Microimmunoelectrophoresis was performed on microscope slides in 1% Ionagar (Oxoid) made up in the appropriate buffer, in the same electrophoresis tank. Agar-gel immunodiffusion was performed in the same agar on 2 x 3-in. microscope slides. These gels, and the microimmunoelectrophoresis gels were stored in a moist atmosphere at room temperature and inspected at intervals over 1--4 days. RESULTS
Immunological reactions of haemocyanins As was to be expected f r o m previous reports (Garvey et al., 1967) M . trunculus haemoeyanin, like other haemocyanins, was a good antigen in rabbits. All five rabbits produced a strong antibody to haemocyanin after the immunization schedule described above. I n agar-gel diffusion a single precipitation line was produced with M . trunculus haemocyanin, and the corresponding apohaemoeyanin reacted similarly. O f the other haemoeyanins tested, that of M . brandaris gave a reaction of partial identity (Fig. 1), while those of C. vulgatum and E. cornea failed to react. I n microimmunoelectrophoresis M . truneulus haemocyanin, as well as the corresponding apohaemocyanin, gave a single precipitation are at all p H values tested between 6-0 and 9-6. T h e mobilities of haemoeyanin and apohaemocyanin appeared to be identical. Some small variations in the pattern obtained were observed depending on the age of the haemoeyanin preparation and on its previous
MUREX TRUNCULUS HAEMOCYANIN
347
treatment. Thus, fresh M . trunculus haemolymph or freshly purified haemocyanin gave a single sharp arc, whereas haemocyanin that had been lyophilized in the presence of sucrose (Heirwegh et al., 1959) and then stored for several months, showed small spurs at either end of the precipitation arc. In all cases however this effect was very small. Photooxidized haemocyanin reacted with the antiserum (Fig. 2D, E) but had a lower mobility than native haemocyanin. Cellulose acetate membrane electrophoresis
Haemocyanin was dialysed against buffers of pH values from 5.4 to 10.8, and then electrophoresis was performed in the same buffer. At pH values below about
All
0
•
!
Fio. 3. Cellulose acetate membrane electrophoresis of M. trunculus haemocyanin at different pH values (tracing). A, 0"1 M acetate, pH 5"5, 100 rain; B, 0.04 M veronal, pH 7"3, 80 rain; C, 0"04 M veronal, pH 8.3, 90 min; D, 0.04 M veronal, pH 9.1, 90 rain; E, 0"2 M glycine, pH 10"4, 90 min. 7.3 a single band was observed, whereas at pH values of 8-3 and above, a number of bands, usually four to six, was always obtained. As Fig. 3 shows, the most common pattern was two strong bands, preceded and followed by weaker bands. In all cases movement was towards the anode. Of the other haemocyanins tested, M . brandaris haemocyanin gave a single band at pH 7.0 which moved slightly more slowly than that of M . trunculus (ratio of mobilities, 1 : 0.93), and three to four bands at pH 9.2. In the latter case a very similar pattern to that produced by M . trunculus haemocyanin was observed. E. cornea haemocyanin similarly produced one band at pH 7.0 and three bands at pH 9.2. At pH 7.0 apohaemocyanin from M . trunculus produced a single band of about the same mobility as native haemocyanin, but there was a faint smear extending back to the origin. At pH 8.6 apohaemocyanin produced two to three diffuse bands, in about the same positions as the bands produced by native haemocyanin, but
348
E.J.
WOOD,
CATHERINEM.
SALISBURY,
N. FORMOSAANDW. H. BANNISTER
again there was streaking back to the origin. Photooxidized haemocyanin produced a smear extending from the origin at all pH values, and no bands could be identified.
Effect of Ca 2+ and Mg 2+ on the stability of haemocyanin In the experiments described above, the observed behaviour is presumably an expression of the tendency of haemocyanins to dissociate into subunits depending on the pH (see Discussion). In these experiments the haemocyanin or haemolymph had been dialysed against 0.1 M phosphate buffer, and was then dialysed against the buffer to be used during electrophoresis. The results therefore show the electrophoretic behaviour of haemocyanin in the calcium- and magnesium-free state. It is known that calcium and magnesium ions are capable of stabilizing haemocyanins from other species against this process of dissociation (Elliott & van Baelen, 1965 ; van Holde & Cohen, 1964). For example, whereas in the absence of Ca 2+ or Mg z+, Helix pomatia haemocyanin starts to dissociate into subunits at pH 7-4-8.1, in the presence of either of these ions, dissociation does not occur until a pH above 9 is reached (Lontie & Witters, 1966; Lontie et al., 1962). Experiments were therefore performed in which haemocyanin was dialysed overnight at 4°C against buffers at various pH values which contained calcium or magnesium salts, and then subjected to electrophoresis in the same buffer. The results are set out in Table 1. Both Ca 2+ and Nlg 2+ at 10 mM concentration extended the stability TABLE 1--EFFECT OF Ca AND Mg
IONS ON M . Trunculus HAEMOCYANIN IN CELLULOSE ACETATE MEMBRANE ELECTROPHORESIS
Dialysis and electrophoresis
Result
pH 7-0 phosphate pH 7"0 phosphate + 10 mM Ca2+ pH 8"15 veronal pH 8"15 veronal + 10 mM Ca2+ pH 8"15 veronal + 10 mM Mg~+ pH 9"2 veronal pH 9"2 veronal+ 10 mM Ca2+ pH 9.2 veronal + 10 rnM Mg2+ pH 9-2 veronal + 100 mM Mg2+
1 band 1 band 4-5 bands 1 band 1 band 4-5 bands lband 3 bands 1 band
region of M. trunculus haemocyanin to pH 8.15 so that a single band was produced. At pH 9.2, Mg ~+ appeared to be less effective in stabilizing the macromolecular structure than Ca ~+, since 10 mM Ca z+, but 100 mM Mg ~+ were required to give a single band at this pH. So far we have been unable to reverse the process of dissociation into subunits in the absence of Mg 2+ or Ca 2+. If haemocyanin was brought to pH 9.2 in the absence of these ions, when it produced four to five bands in electrophoresis, and then dialysed at pH 7.0 either in the presence or absence of calcium or magnesium salts, four to five bands were still produced in electrophoresis at pH 7"0.
M U R E X TRUNCULUS HA.EM0CYANIN
349
No evidence of any heterogeneity was found in microimmunoelectrophoresis under normal conditions over the pH range 6.0-9.6. However, since the agar used contained 0.28% Ca and 0.32% Mg (The Oxoid Manual), it seemed likely that there was enough present of either of these ions to prevent dissociation of haemocyanin in the alkaline pH range. To obviate any effect of these ions, experiments were performed with buffers containing 50 mM EDTA. At pH 6"4 (Fig. 2G) a single precipitation arc formed, but at pH 8.15 (Fig. 2F) two arcs were produced indicating the existence of two species of different mobility and antigenic composition under these conditions. DISCUSSION The present work demonstrates that M. truneulus haemocyanin, like other haemocyanins, is a good antigen, that it reacts in immunodiffusion with a specific antiserum to give a single precipitin line and that in immunoelectrophoresis at pH values below 7"4 in the presence of Ca ~+ or Mg 2+, it gives a single precipitation arc. Furthermore, lyophilization in the presence of sucrose resulted in very small changes as judged by these immunological criteria. The rabbit antiserum to M. trunculus haemocyanin gave a reaction of partial identity with the haemocyanin of the related species M. brandaris, but did not react with the haemocyanins from two other gastropods, E. cornea and C. vulgatum. Malley et al. (1965) demonstrated that the haemocyanin of the giant keyhole limpet (Megathura crenulata) and that of the horseshoe crab (Limulus polyhemus) had no common antigenic determinants. Similarly, Parisi et al. (1962) found that Homarus, Eriphia and Xiphosura haemocyanins had no common determinants. The apohaemocyanin prepared from M. trunculus haemocyanin by treatment with cyanide reacted identically to native haemocyanin. In a study of some of the immunological properties of haemocyanins, Ghiretti's group (Parisi et al., 1962) found that fresh Octopus haemocyanin gave a single sharp precipitation line with its antiserum in immunoelectrophoresis, but that the corresponding apohaemocyanin produced a diffuse arc. They also showed that the precipitation arc produced by apohaemocyanin did not contain copper, and thus did not represent a small amount of contaminating native haemocyanin. It seems therefore that removal of copper by means of cyanide, particularly in the case of M. trunculus haemocyanin, causes minimal changes in the conformation of the protein, which are insufficient to affect the antigenic sites. It is notable that photooxidation of the histidine (and probably other) residues of haemocyanin does not destroy the antigenic reaction. This is similar to the finding of Finger & Schaeg (1967), that complete destruction of all the histidine of insulin left a considerable amount of antigenicity. That the photooxidized insulin and haemocyanin had altered electrophoretic mobilities was to be expected from the destruction or modification of polar residues. Here again one may conclude that destruction of histidine residues in these proteins by means of photooxidation in the presence of methylene blue, does not lead to gross changes in conformation. This is important in the case of haemocyanin, since photooxidation causes loss of
350
E.J. WOOD, CATHERINEM. SALISBURY,N. FORMOSAAND W. H. BANNISTER
copper (Wood & Bannister, 1968), while it is known at the same time that denaturing agents (acids, alcohol, urea) can also cause loss of copper, simply by disrupting the conformation of the protein (see Ghiretti, 1962; Griffd & Lontie, 1961). Previous studies of the reaction of haemocyanin and antiserum in immunoelectrophoresis and in immunodiffusion appear to have taken no account of the calcium and magnesium content of the agar. Parisi et al. (1962) used 1% agar of unspecified origin in a pH 8.4 veronal buffer, and Malley et al. (1965) used 0.85% Ionagar No. 2 (Consolidated Laboratories, Inc.) in pH 8"6 borate buffer. In view of the present findings in experiments where E D T A was included in the buffer, it is perhaps fortuitous that single precipitation arcs were obtained in this previous work, especially as the pH values were beyond the stability zones of some of the haemocyanins. The alternative possibility is that the haemocyanins were completely dissociated into the smallest subunits under these conditions. This is probably less likely, although it does depend on the stability region of the haemocyanin in question. The present finding with cellulose acetate membrane electrophoresis has demonstrated that M. trunculus haemocyanin is heterogeneous in electrophoresis at certain pH values depending upon the presence or absence of calcium or magnesium (Table 1). It seems probable that this may be attributed to subunit formation and one may conclude that the subunits formed are not identical. It is known from ultracentrifuge studies (R. Lontie, personal communication) that M. trunculus haemocyanin splits into half, and then eighth-to-twelfth molecules with increasing pH. However, since between four and six bands were obtained in electrophoresis at pH values above 7.3, it appears likely that either two dissimilar half molecules are produced, or that the smaller subunits are dissimilar, or both. Since a single component appears in the ultracentrifuge at pH values greater than about 8.4 (R. Lontie, personal communication), it seems that small subunits of equal size but of different charge are produced. In cellulose acetate membrane electrophoresis Elliott & van Baelen (1965) found that the haemocyanin from the Congo river snail (Pila le@oldvillensis) gave a single band at pH 5.47, two bands at pH 7.25, three to four bands at pH 8.35, and a single band at pH 9.05. They concluded that the bands obtained represented subunits, and they confirmed this by light-scattering experiments in which the molecular weight was determined as a function of pH. In the buffer of the highest pH (9.05), therefore, a single small subunit exists in Pila haemocyanin. We have been unable to obtain a single band at any pH above 7.3 with M. trunculus haemocyanin. Even in experiments at pH 10.8 several bands continued to appear. REFERENCES
BANNISTERW. H., BANNISTERJ. V. & MICALLEFH. (1966) Purification of hemocyanin from hemolymph by adsorption to calcium phosphate. Experientia 22, 626-627. COHENL. B. & VANHOLDEK. E. (1964) Physical studies of hemocyanins--II. A comparison of the hemocyanin and apohemocyanin of Loligo pealei. Biochemistry 3, 1809-1813. ELmOTT F. G. & VANBAELENH. (1965) Poids moldculaire et zone de stabilitd de l'hdmocyanine de Pila leopoldvillensis. Bull. Soc. Chin. biol. 47, 1979-1986.
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F ~ L , CDEz-MoR~ H., VAN BRUGGEN E. F. G. & OHTSUKI M. (1966) Macromolecular organization of hemocyanins and apohemocyanins as revealed by electron microscopy. jT. Molec. Biol. 16, 191-207. FINGER H. & SCHA~GW. (1967) Die Antigeni~t des photooxidierten Insulins. Biochim. biophys. Acta 140, 532-534. GARWYJ. S., CAMPBELLD. H. & DAS M. L. (1967) Urinary excretion of foreign antigens and RNA following primary and secondary injections of antigens, ft. exp. Med. 125, 111-126. GHIR~tTI F. (1962) Hemerythrin and hemocyanin. In Oxygenases (Edited by HAYAISHIO.), pp. 517--553. Academic Press, New York. GHIlmTTI F. (1966) Molluscan hemocyanins. In Physiology of Mollusca (Edited by WILBUR K. M. & YONGEC. M.), Vol. 2, pp. 233-248. Academic Press, New York. GHImn~rI-MAGALDXA. & NuZZOLOC. (1965) Thiol groups in hemocyanins. Comp. Biochem. Physiol. 16, 249-252. GHIRETTI-MAOALDIA., NUZZOLOC. & GHII~TTI F. (1966) Chemical studies on hemocyanins - - I . Amino acid composition. Biochemistry 5, 1943-1951. G~IFF[ M. & LONTm R. (1961) L'action de l'ur~e sur la liason du cuivre dans l'h6mocyanine d'Helix pomatia. Archs int. Physiol. Biochim. 69, 594--595. HEXRWEGHK., BORGINONH. & LONTm R. (1959) Sur la possibilit~ de lyophiliser les h~mocyanines d'Helix pomatia en pr6sence de saccharose. Archs int. Physiol. Biochim. 67, 514--516. LONTm R., BRAUNSG., COOReMANH. & VANCLEFA. (1962) Distribution of c~- and flhemocyanins in the blood ofHelixpomatia..drchs Biochem. Biophys. Suppl. 1,295-300. LONTIER. & WITTERSR. (1966) Helixpomatia hemocyanins. In The Biochemistry of Copper (Edited by I~mACHJ., AISF~ P. & BLUMBERGW. E.), pp. 455--463. Academic Press, New York. MALLEYA., SAHAA. & HALLIDAYW. J. (1965) Immunochemical studies of haemocyanins from the giant keyhole limpet (Megathura crenulata) and the horseshoe crab (Limulus polyhemus). .7. Immunol. 95, 141-147. PARXSIV., NARDXG., GHIRETTIF. & GHIRET'rx-MACALDXA. (1962) Ricerche sulle emocianine - - I I I . Proprieta' immunologiche. Boll. Soc. ital. Biol. sper. 38, 1848-1851. PETERSONR. E. & BOLLIERM. E. (1955) Spectrophotometrlc determination of serum copper with biscyclohexanoneoxalyldihydrazone. Analyt.Chem. 27, 1195-1197. VAN HOLDEK. E. & COHENL. B. (1964-) Physical studies of hemocyanins--I. Characterization and subunit structure of Loligo pealei hemocyanin. Biochemistry 3, 1803-1808. WOOD E. J. & BANNISTERW. H. (1968) The effect of photooxidation and histidine reagents on Murex trunculus haemocyanin. Biochim. biophys. Acta 154, 10-16.